The erythropoietin receptor (EpoR) is the primary regulator of mammalian erythropoiesis. It lacks intrinsic catalytic activity and relies on Janus Kinase 2 (JAK2) for signal transduction. EpoR dimerization in response to Epo activates JAK2 kinase activity, which in turn phosphorylates tyrosine residues in the EpoR cytoplasmic domain. These phospho-tyrosines recruit signaling effectors to trigger signal transduction. Aberrant activation of the EpoR or JAK2 can lead to leukemia. Our results show that JAK2 is an essential subunit of the EpoR and that the EpoR/JAK2 complex is a functional entity. Little is known about the biochemical structure of the EpoR/JAK2 complex, or how this complex becomes activated upon ligand stimulation. Moreover, results from mutant EpoR knock-in animals suggest that binding of downstream effectors to phospho-tyrosines in the EpoR cytoplasmic domain is not essential for Epo-induced erythropoiesis. We therefore hypothesize that novel JAK2 substrates critical for erythropoiesis exist downstream of the EpoR/JAK2 complex. This proposal aims to delineate the mechanisms regulating the key Epo signaling entity, the EpoR/JAK2 complex, and to characterize novel JAK2 substrates that are vital to EpoR function in erythropoiesis. The specific aims are to: 1. Characterize the EpoR/JAK2 complex. We showed that the N-terminal domain of JAK2, consisting of a PERM and an SH2 domain, is necessary and sufficient for EpoR binding and expression at the cell surface. We will characterize the interaction between the PERM and the SH2 domain that is required for proper JAK2 function. We will also use a novel "protein footprinting" method based on protease sensitivity to map the global interacting surface between the EpoR and the JAK2 N-terminal domain. 2. Determine the molecular mechanism of JAK2 activation. We will identify and characterize specific residues in JAK2 that are required to regulate its activity. Our results identified four EpoR residues essential for JAK2 activation, three of which define a motif conserved among cytokine receptors. We will identify the JAK2 contact sites of these key EpoR residues using chemical protection and cross-linking. 3. Identify and characterize novel JAK2 substrates essential for EpoR function. We will identify novel JAK2 substrates by in vivo immunoprecipitation-mass spectrometry and in vitro genome-wide expression screen. These substrates will be characterized for their roles in erythropoiesis using retrovirus-mediated overexpression and siRNA-mediated depletion in cell lines and in primary erythroid progenitor cells. Information gained from these studies will lead to a more comprehensive understanding of how EpoR signaling regulates erythropoiesis and how oncogenic EpoR and JAK2 activation induces leukemia.